Probing α₄βδ GABA_AReceptor Heterogeneity: Differential Regional Effects of a Functionally Selective α₄β₁δ/α₄β₃δ Receptor Agonist on Tonic and Phasic Inhibition in Rat Brain

Abstract

In the present study, the orthosteric GABA A receptor (GABA A R) ligand 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (Thio-THIP) was found to possess a highly interesting functional profile at recombinant human GABA A Rs and native rat GABA A Rs. Whereas Thio-THIP displayed weak antagonist activity at ␣ 1,2,5 ␤ 2,3 ␥ 2S and 1 GABA A Rs and partial agonism at ␣ 6 ␤ 2,3 ␦ GABA A Rs expressed in Xenopus oocytes, the pronounced agonism exhibited by the compound at ␣ 4 ␤ 1 ␦ and ␣ 4 ␤ 3 ␦ GABA A Rs was contrasted by its negligible activity at the ␣ 4 ␤ 2 ␦ subtype. To elucidate to which extent this in vitro profile translated into functionality at native GABA A Rs, we assessed the effects of 100 M Thio-THIP at synaptic and extrasynaptic receptors in principal cells of four different brain regions by slice electrophysiology. In concordance with its ␣ 6 ␤ 2,3 ␦ agonism, Thio-THIP evoked robust currents through extrasynaptic GABA A Rs in cerebellar granule cells. In contrast, the compound did not elicit significant currents in dentate gyrus granule cells or in striatal medium spiny neurons (MSNs), indicating predominant expression of extrasynaptic ␣ 4 ␤ 2 ␦ receptors in these cells. Interestingly, Thio-THIP evoked differential degrees of currents in ventrobasal thalamus neurons, a diversity that could arise from differential expression of extrasynaptic ␣ 4 ␤␦ subtypes in the cells. Finally, whereas 100 M Thio-THIP did not affect the synaptic currents in ventrobasal thalamus neurons or striatal MSNs, it reduced the current amplitudes recorded from dentate gyrus granule cells, most likely by targeting perisynaptic ␣ 4 ␤␦ receptors expressed at distal dendrites of these cells. Being the first published ligand capable of discriminating between ␤ 2 -and ␤ 3 -containing receptor subtypes, Thio-THIP could be a valuable tool in explorations of native ␣ 4 ␤␦ GABA A Rs.

Functional characterization of GABAA receptor-mediated modulation of cortical neuron network activity in microelectrode array recordings

“…More importantly, THIP is also a more potent agonist at α 4 βδ receptors than at αβγ and αβ receptors, and this selectivity for δ-GABA A Rs within a certain concentration range has made THIP the prototypic pharmacological tool for these receptors [ 42 , 53 – 56 ]. The close structurally related analog Thio-THIP is a functionally subtype-selective α 4 βδ GABA A R ligand, as it acts as a partial agonist at α 4 β 1 δ and α 4 β 3 δ receptors and exhibits negligible agonist activity at α 4 β 2 δ and αβγ GABA A Rs [ 57 ].…”

Functional Characterization of the 1,5-Benzodiazepine Clobazam and Its Major Active Metabolite N-Desmethylclobazam at Human GABAA Receptors Expressed in Xenopus laevis Oocytes

“…The recorded baseline-to-peak current amplitudes were analyzed using Clampfit 10.1 (Axon Instruments, Union City, CA, USA). Analogously to the procedures used in a recent study [ 43 ], the incorporation of the γ 2S subunit into the GABA A Rs assembled at the cell surface of α 1,2,3,5 β 2 γ 2S -expressing oocytes was confirmed on a routinely basis with 100 μM ZnCl 2 [ 46 ]. The presence of δ in cell surface-expressed receptors in α 6 β 2 δ-injected oocytes was confirmed on a routinely basis using the δ-GABA A R selective PAM DS2 (1 μM) [ 47 ].…”

“…Clobazam (synthesized at H. Lundbeck A/S, Denmark), N -desmethylclobazam (from Johnson Matthey Pharma Services, MA, USA), clonazepam (from Lipomed AG, Switzerland), diazepam, and zolpidem (both from Sigma-Aldrich, Denmark) were dissolved in DMSO and diluted in Ringer buffer on the given experimental day. The cDNAs encoding human α 1 , α 2 , α 3 , α 5 , α 6 , β 2 , γ 2S and δ GABA A R subunits were kind gifts from Dr. P J Whiting and Merck, Sharp & Dohme (Harlow, Essex, UK), and they were subcloned into mammalian expression vector pcDNA3.1 (Invitrogen, Denmark) as described previously [ 42 , 43 ].…”

Basic/Translational Development of Forthcoming Opioid- and Nonopioid-Targeted Pain Therapeutics

“…126, 127–129 Subtype specificity may exhibit different effects upon neuronal inhibition in various systems. 130 …”